1
2 An Extensive Program of Periodic Alternative Splicing Linked to Cell Cycle
3 Progression
4
5
6 Authors:
7 Daniel Dominguez1,2, Yi-Hsuan Tsai1,3, Robert Weatheritt 4, Yang Wang1,2,
8 Benjamin J. Blencowe * 4, Zefeng Wang* 1,5
9
10 Affiliations: 11 12 1 Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC 13 2 Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel 14 Hill, NC 15 3 Program in Bioinformatics and Computational Biology, University of North Carolina at Chapel 16 Hill, Chapel Hill, NC, 17 4 Donnelly Centre and Department of Molecular Genetics, University of Toronto, Canada 18 5 Key Lab of Computational Biology, CAS-MPG Partner Institute for Computational Biology, 19 Chinese Academy of Science, Shanghai, China 20
21 * Correspondence to: [email protected]; [email protected]
22 23 24 Abstract
25 Progression through the mitotic cell cycle requires periodic regulation of gene function at
26 the levels of transcription, translation, protein-protein interactions, post-translational
27 modification and degradation. However, the role of alternative splicing (AS) in the temporal
28 control of cell cycle is not well understood. By sequencing the human transcriptome through two
29 continuous cell cycles, we identify ~1,300 genes with cell cycle-dependent AS changes. These
30 genes are significantly enriched in functions linked to cell cycle control, yet they do not
31 significantly overlap genes subject to periodic changes in steady-state transcript levels. Many of
32 the periodically spliced genes are controlled by the SR protein kinase CLK1, whose level
33 undergoes cell cycle-dependent fluctuations via an auto-inhibitory circuit. Disruption of CLK1
34 causes pleiotropic cell cycle defects and loss of proliferation, whereas CLK1 over-expression is
35 associated with various cancers. These results thus reveal a large program of CLK1-regulated
36 periodic AS intimately associated with cell cycle control.
37 38 39 40 Impact statement 41
42 We reveal extensive periodic regulation of alternative splicing during the cell cycle in
43 genes linked to cell cycle functions, and involving an auto-inhibitory mechanism employing the
44 SR protein kinase CLK1.
2 45 Introduction 46 47 Alternative splicing (AS) is a critical step of gene regulation that greatly expands
48 proteomic diversity. Nearly all (>90%) human genes undergo AS and a substantial fraction of
49 the resulting isoforms are thought to have distinct functions (Pan et al., 2008; Wang et al., 2008).
50 AS is tightly controlled, and its mis-regulation is a common cause of human diseases (Wang and
51 Cooper, 2007). Generally, AS is regulated by cis-acting splicing regulatory elements that recruit
52 trans-acting splicing factors to promote or inhibit splicing (Matera and Wang, 2014; Wang and
53 Burge, 2008). Alterations in splicing factor expression have been observed in many cancers and
54 are thought to activate cancer-specific splicing programs that control cell cycle progression,
55 cellular proliferation and migration (David and Manley, 2010; Oltean and Bates, 2014).
56 Consistent with these findings, several splicing factors function as oncogenes or tumor
57 suppressors (Karni et al., 2007; Wang et al., 2014), and cancer-specific splicing alterations often
58 affect genes that function in cell cycle control (Tsai et al., 2015).
59 Progression through the mitotic cell cycle requires periodic regulation of gene function
60 that is primarily achieved through coordination of protein levels with specific cell cycle stages
61 (Harashima et al., 2013; Vermeulen et al., 2003). This temporal coordination enables timely
62 control of molecular events that ensure accurate chromatin duplication and daughter cell
63 segregation. Periodic gene function is conventionally thought to be achieved through stage-
64 dependent gene transcription (Bertoli et al., 2013), translation (Grabek et al., 2015), protein-
65 protein interactions (Satyanarayana and Kaldis, 2009), post-translational protein modifications,
66 and ubiquitin-dependent protein degradation (Mocciaro and Rape, 2012). Although AS is one of
67 the most widespread mechanisms involved in gene regulation, the relationship between the
68 global coordination of AS and the cell cycle has not been investigated.
3 69 Major families of splicing factors include the Serine-Arginine rich proteins (SR) proteins
70 and the heterogeneous nuclear ribonucleoproteins (hnRNPs), whose levels and activities vary
71 across cell types. SR proteins generally contain one or two RNA recognition motifs (RRMs) and
72 a domain rich in alternating Arg and Ser residues (RS domain). Generally, RRM domains confer
73 RNA binding specificity while the RS domain mediates protein-protein and protein-RNA
74 interactions to affect splicing (Long and Caceres, 2009; Zhou and Fu, 2013). Post-translational
75 modifications of SR proteins, most notably phosphorylation, modulate their splicing regulatory
76 capacity by altering protein localization, stability or activity (Gui et al., 1994; Lai et al., 2003;
77 Prasad et al., 1999; Shin and Manley, 2002). Dynamic changes in SR protein phosphorylation
78 have been detected after DNA damage (Edmond et al., 2011; Leva et al., 2012) and during the
79 cell cycle (Gui et al., 1994; Shin and Manley, 2002), suggesting that regulation of AS may have
80 important roles in cell cycle control. However, the functional consequences of SR protein
81 (de)phosphorylation during the cell cycle are largely unclear.
82 Through a global-scale analysis of the human transcriptome at single-nucleotide
83 resolution through two continuous cell cycles, we have identified widespread periodic changes in
84 AS that are coordinated with specific stages of the cell cycle. These periodic AS events belong to
85 a set of genes that is largely separate from the set of genes periodically regulated during the cell
86 cycle at the transcript level, yet the AS regulated set is significantly enriched in cell cycle-
87 associated functions. We further demonstrate that a significant fraction of the periodic AS events
88 is regulated by the SR protein kinase, CLK1, and that CLK itself is also subject to cell cycle-
89 dependent regulation. Moreover, inhibition or depletion of CLK1 causes pleiotropic defects in
90 mitosis that lead to cell death or G1/S arrest, suggesting that the temporal regulation of splicing
91 by CLK1 is critical for cell cycle progression. The discovery of periodic AS thus reveals a
4 92 widespread yet previously underappreciated mechanism for the regulation of gene function
93 during the cell cycle.
94
95 Results
96 Alternative splicing is coordinated with different cell cycle phases
97 To systematically investigate the regulation of AS during the cell cycle, we performed an
98 RNA-Seq analysis of synchronously dividing cells using a total of 2.3 billion reads generated
99 across all stages (G1, S, G2 and M) of two complete rounds of the cell cycle (Figure 1-figure
100 supplement 1A). To maximize the detection of regulated AS events, we used the complementary
101 analysis pipelines, MISO and VAST-TOOLS (Katz et al., 2010; Irimia et al., 2014). These
102 pipelines have different detection specificities and employ partially overlapping reference sets of
103 annotated AS events, and therefore afford a more comprehensive analysis when employed
104 together. Both pipelines were used to determine PSI (the percent of transcript with an exon
105 spliced in) and PIR (the percent of transcripts with an intron retained). Alternative exons
106 detected by both pipelines had highly correlated PSI values (Figure 1-figure supplement 1G; see
107 below). Consistent with previous results (Bar-Joseph et al., 2008; Whitfield et al., 2002),
108 transcripts from approximately 14.2% (1,182) of expressed genes displayed periodic differences
109 in steady-state levels between two or more cell cycle stages (see below). Remarkably, 15.6%
110 (1,293) of expressed genes also contained 1,747 periodically-regulated AS events, among a total
111 of ~40,000 detected splicing events (FDR < 2.5%; Figure 1A and Figure 1-figure supplement
112 1B, 1C, 1D).
113 Importantly, as has been observed previously for AS regulatory networks (Pan et al.,
114 2004), the majority of genes with periodic AS events did not overlap those with periodic steady-
5 115 state changes in mRNA expression. This indicates that genes with periodic changes in AS and
116 transcript levels are largely independently regulated during the cell cycle (Figure 1B). Further
117 supporting this conclusion, we did not observe a significant correlation (positive or negative)
118 between exon PSI values and mRNA expression levels for genes with both periodic expression
119 and periodic exon skipping (data not shown). A gene ontology (GO) analysis reveals that genes
120 with periodic AS, like those with periodic transcript level changes (Bar-Joseph et al., 2008;
121 Whitfield et al., 2002), are significantly enriched in cell cycle-related functional categories,
122 including M-phase, nuclear division and DNA metabolic process (Figure 1C; adjusted P <0.05
123 for all listed categories, FDR <10%) (Supplementary Table1). Similar GO enrichment results
124 were observed when removing the relatively small fraction (10%) of periodically spliced genes
125 that also display significant mRNA expression changes across the cell cycle (Figure 1C). These
126 results thus reveal that numerous genes not previously linked to the cell cycle, as well as
127 previously defined cell cycle-associated genes thought to be constantly expressed across the cell
128 cycle, are in fact subject to periodic regulation at the level of AS (Supplementary File 1 for a full
129 list).
130 Among the different classes of AS analyzed (cassette exons, alternative 5´/3´ splice sites
131 and intron retention [IR]), periodically regulated IR events were over-represented (relative to the
132 background frequency of annotated IR events) by ~2.2 fold whereas periodically regulated
133 cassette exons, represent the next most frequent periodic class of AS (P = 2.2×10-16, Fisher’s
134 exact test, Figure 1-figure supplement 1E). Quantitative RT-PCR assays across different cell
135 cycle stages validated periodic IR events detected by RNA-Seq (Figure 1D). Interestingly, one of
136 these IR events is in transcripts encoding aurora kinase B (AURKB), a critical mitotic factor
137 regulated at the levels of transcription, protein localization, phosphorylation and ubiquitination
6 138 (Carmena et al., 2012; Lens et al., 2010). The AURKB retained intron is predicted to introduce a
139 premature termination codon that elicits mRNA degradation through nonsense mediated decay,
140 and is thus expected to result in reduced levels of AURKB protein. The splicing of the retained
141 intron lags behind changes in the total AURKB mRNA expression (Figure 1E). We
142 computationally corrected levels of fully spliced, protein coding AURKB mRNA by taking into
143 account the fraction of intron-retaining (i.e. non-productive) transcripts across the cell cycle
144 stages (Figure 1E). The expression curve for corrected AURKB mRNA levels is substantially
145 different from total AURKB transcript levels, with a shifted peak coinciding with mitosis.
146 Periodically-regulated IR events detected in other genes, including those with known cell cycle
147 functions such as HMG20B and RAD52, are similarly expected to affect the cell cycle timing of
148 mRNA expression (Figure 1A, 1D). Collectively, these results provide evidence that the
149 temporal control of retained intron AS provides an important mechanism for establishing the
150 timing of expression of AURKB mRNA and protein, as well as of the timing of expression of
151 additional genes during the cell cycle.
152
153 The SR protein kinase CLK1 fluctuates during the cell cycle
154 Alternative splicing is generally regulated by the concerted action of multiple cis-
155 elements that recruit cognate splicing factors. Consistently, analysis of our RNA-seq data
156 revealed 96 RNA binding proteins (RBPs) with periodic mRNA expression, including RS
157 domain-containing factors like SRSF2, SRSF8, TRA2A and SRSF6 (Figure 2-figure supplement
158 1, 2A, Table 1). These 96 RBPs were significantly enriched in the GO term “splicing regulation”
159 (adjusted P = 10-4, Figure 2-figure supplement 2 2B), indicating that periodic AS is likely
160 controlled by multiple RBPs. Correlations between these RBPs and periodic splicing events were
7 161 also identified (Figure 2-figure supplement 1, 2C). For example, SRSF2 expression is
162 significantly correlated with the splicing pattern of a retained intron in the SRSF2 transcript.
163 Further supporting a role for these RBPs in controlling periodic splicing was the identification of
164 RNA motifs bound by a subset of periodically expressed RBPs (Figure 2-figure supplement 1,
165 2D). To further examine periodic RBP regulation during cell cycle, we measured the abundance
166 of known splicing regulatory proteins at different stages of the cell cycle by immunoblotting
167 (Figure 2A). Among the proteins analyzed, CDC-like kinase 1 (CLK1), an important regulator of
168 the Ser/Arg (SR) repeat family of splicing regulators, displayed the strongest cyclic expression
169 peaking at the G2/M phase (Figure 2A, 2B), consistent with the results of a recent mass-
170 spectrometry-based screen for cycling proteins (Ly et al., 2014). CLK1 is one of four human
171 CLK paralogs (CLK1-4) and is known to regulate AS via altering the phosphorylation status of
172 multiple SR proteins (Duncan et al., 1997; Jiang et al., 2009; Ninomiya et al., 2011; Prasad et al.,
173 1999). Notably, the levels of other detectable CLK paralogs, as well as members of another SR
174 protein kinase, SRPK1, did not change significantly at the level of RNA and/or protein during
175 the cell cycle (Figure 2B and Supplementary File 1).
176 Given that both CLK1 protein levels and known CLK1 substrates are periodically
177 expressed, we decided to further investigate the role of CLK1 in the context of cell cycle. The
178 levels of total CLK1 mRNA, as well as the levels of specific CLK1 splice variants, did not
179 change significantly during the cell cycle (Figure 2-figure supplement 2, 1A, 1B) indicating that
180 periodic expression of CLK1 is controlled at the level of protein translation and/or turnover.
181 Consistent with this, an exogenously expressed CLK1 protein displayed cell cycle-dependent
182 fluctuations similar to those observed for endogenous CLK1 protein (Figure 2B). Moreover,
183 CLK1 was rapidly degraded upon inhibition of translation by cycloheximide, and this effect was
8 184 reversed by co-treatment with the proteasome inhibitor MG132 (Figure 2-figure supplement 2
185 1C). Additionally, polyubiquitination of Flag-tagged CLK1 was detected following
186 immunoprecipitation with anti-Flag antibody from cells treated with MG132 (Figure 2–figure
187 supplement 2 1D). These data suggest that the levels of CLK1 protein are controlled by
188 ubiquitin-mediated degradation in a cell cycle-dependent manner.
189 Periodically regulated protein levels are often controlled through negative feedback
190 circuits involving auto-regulatory loops. CLK1 has been reported to auto-phosphorylate on
191 several residues (Ben-David et al., 1991). To investigate whether auto-phosphorylation of CLK1
192 affects its periodic regulation, we tested whether blocking its kinase activity affects its stability.
193 Inhibition of CLK1 kinase activity using a selective inhibitor, TG003 (Muraki et al., 2004),
194 markedly stabilizes both endogenous and exogenously expressed CLK1 proteins (Figure 2C).
195 Moreover, activity-dependent destabilization of CLK1 was observed with a wild type (WT)
196 protein, but not with a catalytically inactive (KD) mutant (Figure 2C, left panel). We further
197 observe that WT CLK1 is rapidly degraded upon cycloheximide treatment, whereas the KD
198 mutant is more stable (Figure 2–figure supplement 1C). We next tested whether CLK1 activity
199 is sufficient to trigger its own degradation by co-expressing KD CLK1 with increasing amounts
200 of WT CLK1. As expected, increasing amounts of WT CLK1 reduces levels of KD CLK1
201 (Figure 2D). Consistent with these results, WT CLK1 is more highly polyubiquitinated compared
202 to the KD mutant (Figure 2E, compare lanes 2 to 4), and treatment with TG003 reduces
203 polyubiquitination levels (Figure 2E, lane 2 vs. 3 and lane 4 vs. 5). Decreased polyubiquitination
204 of WT CLK1 is more prevalent than is apparent upon TG003 treatment, as CLK1 is stabilized by
205 TG003 inhibition and thus more total Flag-CLK1 is immunoprecipitated (Figure 2E). To further
206 examine whether this auto-feedback loop is required for changes in CLK1 protein levels during
9 207 the cell cycle, we treated synchronized cells with TG003 (or DMSO as a control) and measured
208 CLK1 protein levels. We observed that CLK1 inhibition prevents its turnover after the G2/M
209 phase for both endogenous and exogenously expressed kinases (Figure 2F and Figure 2–figure
210 supplement 1E). Taken together, these results provide strong evidence that CLK1 protein levels
211 are controlled by ubiquitin-mediated proteolysis in a cell cycle stage-specific manner, and that an
212 activity-dependent negative feedback loop is required for this periodic regulation. These results
213 further suggest that changes in the levels of CLK1 could account for many of the periodically
214 regulated AS transitions we have detected during the cell cycle.
215
216 CLK1 regulates AS events in genes with critical roles in cell cycle control
217 RNA-Seq analysis of cells treated with TG003 revealed 892 AS events (in 665 genes)
218 that significantly change after CLK1 inhibition (Figure 3A), including known CLK1-regulated
219 splicing events (e.g. exon 4 of CLK1 (Duncan et al., 1997)). It is worth noting that TG003 can
220 also inhibit CLK4 (although to a lesser extent than inhibition of CLK1). However, RNAi of
221 CLK1 is sufficient to recapitulate the phenotype of CLK1/4 inhibition (see below and Figure 4)
222 (Fedorov et al., 2011; Muraki et al., 2004). Intron retention and cassette exons are the most
223 overrepresented types of AS affected by TG003 (Figure 3A). Most (70%) of the CLK1-regulated
224 exons display increased skipping upon CLK1 inhibition, whereas 87% of CLK1-regulated
225 introns show increased retention (Figure 3-figure supplement 1B and 1C), consistent with a
226 recently reported role for CLK1 in the regulation of retained introns (Boutz et al., 2015). Of nine
227 analyzed TG003-affected AS events detected by RNA-Seq analysis, all were validated by semi-
228 quantitative RT-PCR assays (Figure 3B). These observations indicate that CLK1 inhibition
229 mainly suppresses splicing, consistent with a general requirement for phosphorylation of SR
10 230 proteins to promote splicing activity (Irimia et al., 2014; Prasad et al., 1999; Tsai et al., 2015).
231 Importantly, there is a significant overlap between genes with cell cycle periodic AS events
232 (Figure 1) and those with CLK1-regulated AS events, involving 156 genes (P = 8.5×10-10, hyper-
233 geometric test). In contrast, consistent with the results in Figure 1, we do not observe a
234 significant overlap between genes containing CLK1-regulated AS events and periodically
235 expressed genes. These results thus support a widespread and rapidly acting role for CLK1 in
236 controlling cell cycle-regulated AS. Indeed, CLK1 inhibition induces rapid (within 3-6 hours)
237 changes in AS among several analyzed cases (Figure 3-figure supplement 1D).
238 Supporting an important role for CLK1 in cell cycle progression, genes whose AS levels
239 are affected by CLK1 inhibition are significantly enriched in the GO terms cell cycle phase, M-
240 phase, DNA metabolic processes, nuclear division, DNA damage response, and cytokinesis
241 (adjusted P < 0.05 and FDR <20% for all listed GO terms; full list in Supplementary File 2). The
242 affected genes function at various stages of cell cycle including the G1/S transition (Figure 3C).
243 Mitotic processes were, however, associated with the largest number of CLK1-target genes with
244 AS changes and included examples that function in centriole duplication (CEP70, CEP120,
245 CEP290, CEP68, CDK5RAP2), metaphase and anaphase (e.g. CENPK, CENPE, CENPN), and
246 cytokinesis (e.g. SEPT2, SEPT10, ANLN) (additional examples in Figure 3C).
247 To further investigate the functional consequence of CLK1-dependent AS, we selected
248 two examples in genes that have important roles in the cell cycle: checkpoint kinase 2 (CHEK2),
249 a tumor suppressor that controls the cellular response to DNA damage and cell cycle entry
250 (Paronetto et al., 2011; Staalesen et al., 2004), and centromere-associated protein E (CENPE), a
251 kinetochore-associated motor protein that functions in chromosome alignment and segregation
252 during mitosis (Kim et al., 2008). We detected a TG003 dose-dependent increase in CHEK2
11 253 exon 9 inclusion, whereas overexpression of WT CLK1 induced exon 9 skipping, an event that
254 removes the CHEK2 kinase domain (Figure 3D). Expression of WT CLK1 in the presence of
255 TG003, or a catalytically inactive CLK1, had little to no effect on the splicing of this exon
256 (Figure 3D, bottom panels), indicating that the catalytic activity of CLK1 is essential for
257 regulating CHEK2 AS. Over-expression of the SR protein splicing regulator SRSF1, a known
258 target of CLK1 (Prasad et al., 1999), had a similar effect as over-expression of CLK1, resulting
259 in CHEK2 exon 9 skipping, whereas knockdown of SRSF1 had the opposite effect (Figure 3D,
260 right panel). Furthermore, the activation of CHEK2 requires homodimerization (Shen et al.,
261 2004), and we observe that the CHEK2 isoform lacking exon 9 still interacts with full-length
262 protein (Figure 3-figure supplement 1E), suggesting that this CLK1-regulated isoform may
263 function in a dominant-negative manner to attenuate CHEK2 activity.
264 CENPE is known to be tightly controlled at multiple levels (including transcription,
265 localization, phosphorylation and degradation), and disruption of its regulation leads to
266 pronounced mitotic defects. CENPE AS generates long and short isoforms (Supplementary File
267 2), with the predominant variant being the short isoform that lacks amino acids 1972-2068.
268 Inhibition of CLK1 rapidly shifts CENPE splicing to produce predominantly the long isoform
269 (Figure 3E), and this is accompanied by a reduction in CENPE protein levels during G2/M
270 phase, presumably due to the instability of the long isoform (Figure 3E). These data thus show
271 that CLK1 controls the AS of major cell cycle regulators, and therefore suggest that inhibition of
272 CLK1 may alter cell cycle progression.
273 To investigate this, we next performed an RNA-Seq analysis of synchronized cells at G1
274 and early G2 (when CLK1 accumulates), following inhibition of CLK1 with TG003. As a
275 specificity control, we performed a parallel RNA-Seq analysis using a structurally distinct CLK1
12 276 inhibitor, KHCB-19 (Fedorov et al., 2011). Strikingly, of 1,498 AS events that change between
277 G1 (t=0) and G2 (t=6) ~94% display reduced changes following treatment with the two drugs
278 (Figure 3F), with 65% commonly affected by both drugs, thus supporting an important role for
279 CLK1 in controlling cell-cycle dependent splicing.
280
281 CLK1 is Required for Normal Mitosis and Cell Proliferation
282 Given that CLK1 regulates the AS of many cell cycle factors (Figure 3C), we next
283 examined whether it is necessary for cell cycle progression. Knockdown of CLK1 using shRNAs
284 led to an accumulation of cells with 4N DNA content in multiple cell types, specifically HeLa,
285 H157, and A549 (Figure 4A, Figure 4-figure supplement 1 1A, 1B), and the extent of this
286 accumulation correlated with the degree of knockdown (Figure 4-figure supplement 1 1A). We
287 also observed a significant increase in multi-nucleation, a common consequence of defective
288 chromosome segregation or cytokinesis, following shRNA-knockdown or TG003 inhibition of
289 CLK1 in the treated cells (Figure 4B and Figure 4-figure supplement 1 1C). To visualize the
290 effect of CLK1 inhibition on mitosis at a single cell level, we performed time-lapse high-content
291 microscopy on live cells stably expressing a GFP-histone 2B fusion protein, to track changes in
292 chromatin. TG003-treated cells entered mitosis normally, as measured by nuclear envelope
293 breakdown, but displayed delayed or aberrant cytokinesis, typically resulting in multi-polar
294 divisions, increased time in metaphase, failure to undergo chromatin de-condensation and
295 eventual cell death (Figure 4C and Video 1-5). To further determine at what cell cycle stage
296 CLK1 activity is required, we inhibited CLK1 using TG003 at different time points after early S
297 phase release. Consistent with the imaging data, both control and TG003-treated cells entered
298 mitosis normally, as measured by 4N DNA content. However, inhibition of CLK1 before late S-
13 299 phase impaired progression through mitosis, whereas cells treated 5 hours after early S phase
300 release underwent a round of normal mitotic division, although failed to enter the next cell cycle
301 (Figure 4D and Figure 4-figure supplement 1 1D). These results suggest that the primary defects
302 caused by CLK1 inhibition occur in late S-phase and G2 phase, which is when CLK1 levels
303 normally begin to rise (Figure 2A). This conclusion is further supported by the observation that
304 CLK1-dependent AS targets, such as those detected in HMMR and CENPE, are periodically
305 expressed during cell cycle and peak during G2 and M phase (Figure 4-figure supplement 1 1E).
306 Taken together with the earlier results, these data support an important and multifaceted role for
307 CLK1 in the control of cell cycle progression through its function in the global regulation of
308 periodic AS.
309
310 CLK1 expression and CLK1-regulated AS is altered in kidney cancer
311 The importance of CLK1 for faithful progression through the cell cycle suggests it may
312 play a role the control of cell proliferation in cancer. Supporting this, shRNA knockdown or
313 chemical inhibition of CLK1 with TG003 or KHCB-19 in HeLa cells results in a near complete
314 block in cell proliferation, as measured by anchorage-dependent and -independent colony
315 formation assays (Figure 4E and Figure 4-figure supplement 1F). As mentioned above, CLK1 is
316 likely the primary target of inhibition in these experiments since RNAi of CLK1 recapitulates the
317 phenotype seen with these chemical inhibitors.
318 Using RNA-Seq data from the Cancer Genome Atlas (TCGA) (Cancer Genome Atlas
319 Network, 2013) we observe that CLK1 displays significantly higher expression in 72 kidney
320 tumors compared to matched normal tissue samples (Figure 4F, P = 10-5, Kolmogorov-Smirnov
321 test). Consistently, most CLK1-controlled AS events that are altered in tumors have expected
14 322 splicing changes (Figure 4-figure supplement 2 2A). Furthermore, patients with tumors that have
323 elevated CLK1 expression (i.e. the upper quartile of all samples) have significantly lower
324 survival rates relative to other patients in the comparison group (Figure 4G, P = 0.007). While
325 there was also an increase in CLK2, CLK3 and CLK4 mRNA expression in these tumors, CLK1
326 displayed the highest relative mRNA expression levels compared to CLK2 and CLK3 (Figure 4-
327 figure supplement 2 2B). Levels of the CLK4, which is ~80% identical to CLK1, did not
328 correlate with survival differences despite its increased levels in tumors (Figure 4-figure
329 supplement 2 2C). Consistent with this, CLK1-regulated AS events, as defined by the RNA-Seq
330 analysis in Figure 3, were also altered across multiple tumor types, including breast, colon, lung
331 and liver (Figure 4H and Figure 4-figure supplement 2 2D). These data are further consistent
332 with a multi-faceted role for CLK1 in regulating cell cycle progression, and also suggest that
333 CLK1 contributes to increased cell proliferation in cancer, at least in part through its role in
334 controlling periodic AS.
335
336 Discussion
337 Previous studies have shown that splicing and the cell cycle are intimately connected
338 processes. Indeed, cell cycle division (CDC) loci originally defined in S. cerevisiae, namely cdc5
339 and cdc40, were subsequently shown to encode spliceosomal components (Ben-Yehuda et al.,
340 2000; McDonald et al., 1999). Moreover, genome-wide RNAi screens for new AS regulators of
341 apoptosis genes in human cells revealed that factors involved in cell-cycle control, in addition to
342 RNA processing components, were among the most significantly enriched hits (Moore et al.,
343 2010; Tejedor et al., 2015). An RNAi screen performed in Drosophila cells for genes required
344 for cell-cycle progression identified numerous splicing components(Bjorklund et al., 2006) as
15 345 well as a Drosophila ortholog of CLK kinases, Darkener of apricot Doa (Bettencourt-Dias et al.,
346 2004). In other studies, negative control of splicing during M phase was shown to be dependent
347 on dephosphorylation of the SR family protein, SRSF10 (Shin and Manley, 2002), and the
348 mitotic regulator aurora kinase A (AURKA) was shown to control the AS regulatory activity of
349 SRSF1(Moore et al., 2010). Our study shows for the first time that AS patterns are subject to
350 extensive periodic regulation, in part via a global control mechanism involving cell cycle
351 fluctuations of the SR protein kinase CLK1. At least one likely function of this periodic AS
352 regulation is to control the timing of activation of AURKB (Figure 1), as well as of numerous
353 other key cell cycle factors shown here to be subject to periodic AS. The definition of an
354 extensive, periodically-regulated AS program in the present study thus opens the door to
355 understanding the functions of an additional layer of regulation associated with cell cycle control
356 and cancer.
357 Since many RBPs are found to be periodically expressed (Fig. 4A), it is likely that other
358 aspects of mRNA metabolism are also coordinated with cell cycle stages. For example,
359 differential degradation could potentially contribute to the observed periodic fluctuation of splice
360 isoforms during cell cycle. The degradation of mRNA is closely linked with alternative
361 polyadenylation, which has emerged as a critical mechanism that controls mRNA translation and
362 stability. Generally, shortened 3′ UTRs are found in rapidly dividing cells and more aggressive
363 cancers (Mayr and Bartel, 2009). This study identified 94 cases periodic alternative poly-A site
364 usage (data not shown) in genes known to regulate cell cycle and/or proliferation, including
365 SON, CENPF and EPCAM (Ahn et al., 2011; Bomont et al., 2005; Chaves-Perez et al., 2013). In
366 addition, many mRNAs have recently been found to be translated in a cell cycle dependent
367 fashion (Aviner et al., 2015; Maslon et al., 2014; Stumpf et al., 2013). Interestingly, a fraction of
16 368 periodically translated genes are also periodically spliced, including key regulators of cell cycle
369 (e.g., AURKA, AURKB, TTBK1 and DICER1). This observation is consistent with recent findings
370 that the regulation of AS and translation may be coupled (Sterne-Weiler et al., 2013). In
371 summary, we have demonstrated that AS is subject to extensive temporal regulation during the
372 cell cycle in a manner that appears to be highly integrated with orthogonal layers of cell cycle
373 control. These results thus provide a new perspective on cell cycle regulation that should be
374 taken into consideration when studying this fundamental biological process, both in the context
375 of normal physiology and diseases including cancers.
376
17 377 Methods
378 Cell culture and synchronizations: HeLa (a kind gift from J. Trejo), HEK 293T (from ATCC
379 CRL-3216) and A549 (kind gift from W. Kim) cells were maintained in DMEM (Gibco)
380 medium supplemented with 10% FBS (Gibco). All cells were cultured in humidified incubators
381 with 5% CO2. Cell cycle synchronization was adapted from the protocol of Whitfield et al.
382 (Whitfield et al., 2002); ~ 750,000 log phase HeLa cells were plated in 15 cm dishes in complete
383 media and allowed to attach for 16 hrs, reaching < 30% confluence. Cells were subsequently
384 treated with 2 mM thymidine (Sigma) for a total of 18 hrs, washed 2 times with 1xPBS, and
385 supplemented with fresh complete media for 10 hrs. 2 mM thymidine was subsequently added
386 for a second block of 18 hrs and washed as described previously. Mitotic block was performed
387 by double thymidine arrest (as above) and release in fresh media for 3.5 hrs followed by addition
388 of nocodazole 100 μM (Sigma) for 10 hrs. G1 block was performed by serum starvation for 72
389 hrs in DMEM containing 0.05% FBS. For RNA-Seq, cells (both adherent and detached) were
390 harvested every 1.5 hrs for 30 hrs and frozen immediately for purification of total RNA. To
391 block the activity of CLK1, cells were treated with TG003 (Sigma), KHCB-19 (Tocris). To
392 block activity of the proteasome cells were treated with MG132 (Sigma). Drugs were-suspended
393 in DMSO and added to growing cultures at the indicated concentrations and times.
394 Flow cytometry and cell cycle analysis: Cells were harvested with trypsin treatment, washed 2
395 times in cold 1xPBS and subsequently fixed in 80% ice cold ethanol for at least 4 hrs. Cells were
396 then washed twice with 1xPBS and suspended in propidium iodide/RNase staining buffer (BD
397 Pharmingen, cat # 550825). Cells were analyzed by flow cytometry to count 10,000 cells that
398 satisfied gating criteria. Data collected were analyzed using ModFit software to discern 2N (G1),
399 S-phase, and 4N (G2 and M) composition.
18 400 Mapping and filtering of RNA-Seq data: RNA-Seq reads were mapped to the human genome
401 (build hg19) using the MapSplice informatics tool with default parameters (Wang et al., 2010).
402 The mapped reads were further analyzed with Cufflinks to calculate the level of gene expression
403 with FPKM (Fragments Per Kilobase per Million mapped reads) (Trapnell et al., 2010). The
404 levels of alternatively spliced isoforms were quantified with MISO (Mixture-of-Isoforms)
405 probabilistic framework (Katz et al., 2010) using the annotated AS events for human
406 hg19version 2. The levels of alternatively spliced isoforms were also quantified with VAST-
407 TOOLS using the event annotation as previously described (Irimia et al., 2014). Each AS event
408 was assigned a PSI or PIR value to represent the percent of transcripts with the exon spliced in,
409 or the intron retained, respectively.
410 Identification of periodic AS:
411 For identification of periodic AS raw PSI/PIR values were normalized as:
Φ − Φ (Φ )= Φ
412 where s = 1 to 32,109 for all splicing events; n = 1 to 14 for the 14 samples; Φ is the
413 minimum and Φ is the maximum PSI value among the 14 samples.
414 To identify periodic AS events, normalized gene expression values (normalized FPKM
415 values as ) for the well- known periodic gene, CCNB1, CCNA2, CCNB2, and CENPE, were
416 used as a starting point to subsequently add curves with broader or sharper peaks as well as
417 shifted to left or right, resulting in 7 periodic expression curves that cover all the phases of cell
418 cycle (Figure 1-figure supplement 1A). We term these “ideal seed curves”, which capture
419 intermittent peak times and phase shifts that were not well represented within the initial known
19 420 periodic genes. To identify genes with similar splicing patterns across the cell cycle, we
421 computed the Euclidean Distance of each AS event s to the model seed curves m as follows:
, = | (e )− (Φ )|
422 where m = 1 to 7 for all model seed curves, s = 1 to 32,109 for all AS events.
423 Based on the ranking of distance, a similar cutoff of ≤ 2.75 was set as a minimum
424 requirement for periodic AS. Lastly, we calculated a false discovery rate (FDR) by shuffling PSI
425 values across the 14 time points 10,000 times and calculating how often a random shuffle had a
426 better periodic score than the true periodic score for that event. A maximum FDR of 2.5% was
427 required for a splicing event to be periodic.
428 Heat maps, correlations, GO-term analysis, overlap analysis and statistics: Heat maps,
429 hierarchical clustering, and Pearson correlations were generated using GENE-E
430 (www.broadinstitute.org/cancer/software/GENE-E/). All heat maps shown are row-normalized
431 for presentation purposes. Spearman’s rank correlation with average linkage was used for
432 clustering. DAVID (http://david.abcc.ncifcrf.gov/gene2gene.jsp) was used for all gene ontology
433 enrichment; terms shown are for biological process (GOTERM_BP_FAT). To test for
434 significance in overlap analysis, overlapping genes in two data sets (i.e. TG003-treatment and
435 periodic AS) and a background set of only co-detected events was used (i.e. genes detected in
436 both experiments). Significance of overlapping gene sets was assessed using the hyper-geometric
437 test. For overlap and correlation analysis between VAST-TOOLS and MISO we used two MISO
438 AS event annotations (HG18 and HG19), due to differences in the input annotation files for these
439 two pipelines. To identify the 4,343 overlapping exons in the heatmap, we used MISO hg19v1
20 440 annotations and MISO hg18 annotations, and the VAST-TOOLS annotations. Student’s t-test
441 was used to measure significant in cell cycle defects (multi-nucleation and flow-cytometry) as
442 well as semi-quantitative RT-PCR assessment of splice variants. To identify AS events blocked
443 by TG003 or KHCB19, a Student’s t-test statistic was used. If a change between G1/S (t=0) and
444 G2 (t=6) was significant, but not significant in the presence of inhibitors we consider that event
445 to be blocked. For over-representation of periodic introns, we performed Fisher’s exact test in a
446 2x2 contingency table as compared to skipped exons. For differences in expression of CLK1
447 mRNAs in kidney cancers a Kolmogorov-Smirnov test was performed. For survival differences,
448 the survdiff function in the R survival package was used (as discussed in methods below).
449 Plasmid construction, transfections and RNAi: The expression constructs were generated by
450 cloning the cDNA of CLK1 into pCDNA3 (for transient expression) or pCDH (for stable
451 transfection) backbones with different epitope tags (HA or Flag) at the N- or C-terminus. The
452 Myc-His-Ubiquitin expression vector is a gift from Dr. Gary Johnson’s lab, and the Histone
453 H2B-GFP expression vector is gift from Dr. Angelique Whitehurst’s lab. Plasmid transfections
454 were performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s
455 protocol. The lentiviral vectors of shRNAs were obtained from Addgene in pLKO.1 TRC
456 cloning plasmid through UNC core facility as part of mammalian gene knockdown consortium.
457 Lentiviral infections were performed according to the manufacturer's instruction from System
458 Biosciences (SBI).
459 Semi-quantitative RT-PCR assays for monitoring splicing: Cells transfected with shRNA
460 constructs or treated with TG003 were lysed and total RNA was extracted using the Trizol
461 method (Life Technologies). Purified RNA was treated with 1U of RNAse-free DNAase
462 (Promega) for 1 h at 37ºC and reverse transcribed using random hexamer cDNA preparation kit
21 463 (Applied Biosystem). One-tenth of the RT product was used as the template for PCR
464 amplification (25 cycles of amplification, with a trace amount of Cy5-dCTP in addition to non-
465 fluorescent dNTPs) using gene specific primers listed in Supplementary File 4. The resulting gels
466 were scanned with a Typhoon 8600 Imager (GE Healthcare), and analyzed with ImageQuant 5.2
467 software (Molecular Dynamics/GE Healthcare). Real time PCR was carried out using the SYBR
468 Green kit (Invitrogen) and GAPDH as an internal control.
469 Immunoblotting and immunoprecipitation: Proteins were extracted in lysis buffer (CHAPS
470 1% w/v, 150 mM NaCl, 50 mM MgCl2 with protease inhibitor), resolved by SDS-PAGE and
471 transferred onto PVDF membrane. For immunoprecipitaion experiments to detect ubiquitination,
472 cells were co-transfected with Flag-CLK1 and myc-ubiquitin constructs as above. 36 hrs later,
473 TG003 (20 μM) was added for 18 hrs. 4 hours prior to harvesting, 10 μM of MG132 was added
474 to the media. Proteins were extracted in lysis buffer as above with the addition of NEM.
475 Incubation with EZ-View FLAG Beads (Sigma) was performed for 2 hrs at 4oC. Samples were
476 extensively washed according to the manufacturer’s protocol and subjected to immunoblotting.
477 Antibodies and dilutions are listed in Supplementary File 4.
478 Immunofluorescence and high-content live-cell imaging: For immunofluorescence
479 microscopy, cells were plated on glass coverslips coated with poly-L-Lysine. Cells were then
480 washed twice with 1xPBS, fixed with 4% formaldehyde (Sigma), permeabilized with 0.05%
481 Triton X-100 (Promega) and blocked with 3% BSA (Fisher); all dilutions were made in 1XPBS.
482 For live cell imaging, HeLa cells transduced with Histone H2B-GFP was stably selected as
483 described previously (Cappell et al., 2010). Cells were plated in a 6-well format and treated with
484 2 mM thymidine for 24 hrs, subsequently washed and released in fresh complete medium with or
22 485 without TG003 (20 µM). Cells were imaged using the BD Pathway Microscope with a 10X
486 objective.
487 Colony formation assays: HeLa cells stably producing shRNAs targeting CLK1 or control
488 shRNAs were plated at low density (1,000 cells/6 cm2) in standard culture medium and allowed
489 to proliferate for 9 days. Cells were then fixed and stained with crystal violet at room
490 temperature. The dried plates were used for estimations of colony diameter and number.
491 Kidney Cancer Analysis of CLK1 and Alternative Splicing:
492 RNA-Seq data from the The Cancer Genome Atlas (Ciriello et al., 2013) was processed as
493 previously described (Tsai et al., 2015). Briefly, for mRNA expression, RSEM expression values
494 for the indicated genes were analyzed in 79 paired KIRC (tumor and normal) samples and the
495 paired ks-test was used to test significance. For alternative splicing analysis data from BRCA:
496 Breast invasive carcinoma, COAD: Colorectal adenocarcinoma, KIRC: Kidney renal clear cell
497 carcinoma, LUAD: Lung adenocarcinoma, LUSC: Lung squamous cell carcinoma, LIHC: liver
498 hepatocellular carcinoma were analyzed through the MISO pipeline as described above (Tsai et
499 al., 2015). Relapse-free survival was analyzed using Kaplan Meier plots. All plots and statistical
500 analyses (survdiff) were generated using the R package version 3.1.1 survival function.
501
23 502 Acknowledgements: 503
504 We thank Dr. Jan Prins and Darshan Singh for helpful suggestions in analyzing RNA-Seq
505 data, and Drs. Angelique Whitehurst, William Marzluff and Jean Cook for sharing reagents. We
506 thank Drs. Jun Zhu and Ting Ni for advice and assistance in generating strand-specific RNA
507 library. Dr. Rebecca Sinnott provided assistance in live cell imaging experiments. We thank Drs.
508 William Marzluff, Chris Burge, Jean Cook, Michael Emanuele and Mauro Calabrese for helpful
509 comments on the manuscript. This work is supported by an NIH grant (R01CA158283) and
510 Jefferson-Pilot fellowship to Z.W., and by grants from the Canadian Institutes of Health
511 Research to B.J.B. B.J.B. holds the Banbury Chair of Medical Research at the University of
512 Toronto.
513 514 Author Contributions:
515 D.I.D. and Z.W. conceived the project design, and D.I.D. performed the experiments. Y.-
516 H.T., D.I.D., R.W., Y.W., B.J.B. and Z.W. analyzed data. D.I.D., Z.W. and B.J.B. wrote the
517 paper with input from other authors.
518
519 Disclosure of Potential Conflicts of Interest
520 No potential conflicts of interest to disclose by the authors of this work.
521
522 Supplemental Information:
523 Supplementary File 1. Periodic splicing events identified during the HeLa cell cycle and 524 associated GO terms. Data associated with Figure 1 525 526 Supplementary File 2. AS events altered by CLK1 inhibition (TG003 10μM for 16hrs) in 293T 527 cells and associated GO terms. Data associated with Figure 3. 528
24 529 Supplementary File 3. AS events altered by CLK1 inhibition (TG003 10μM, KHCB19 10μM) in 530 synchronized HeLa cells. Data associated with Figure 3. 531 532 Supplementary File 4. List of PCR primers, shRNA sequences and antibodies used in this study. 533 534 Video 1. Live-cell imaging of control HeLa cells stably expressing a GFP-H2B. Cells were 535 synchronized by single-thymidine block and released and imaged at 10X magnification (every 536 ~15 min) for 960 min. 537 538 Video 2. Live-cell imaging of control HeLa cells stably expressing GFP-H2B. Cells were 539 synchronized by single-thymidine block and released into 1 μM of TG003 and imaged at 10X 540 magnification (every ~15 min) for 960 min. 541 542 Video 3. Zoom of live-cell imaging of control HeLa cells. Data associated with Figure 4D. 543 544 Video 4. Zoom of live-cell imaging of TG003-treated HeLa cells. Data associated with Figure 4 545 546 Video 5. Zoom of live-cell imaging of TG003-treated HeLa cells. Data associated with Figure 4 547
25 548 Main Figure Legends 549 550 Figure 1: Global detection of periodic cell cycle-dependent alternative splicing.
551 A, Heat map representation of periodically spliced events. Row-normalized relative PSI values
552 are shown. Diagram below indicates cell cycle phase.
553 B, Overlap between periodically spliced genes and periodically expressed genes detected by
554 RNA-Seq.
555 C, Heat map representation of enriched Gene Ontology terms shown as log(P-value). Three gene
556 sets were analyzed separately: all genes with periodic AS, genes with periodic AS only, and
557 genes with both periodic AS and periodic expression.
558 D, Real-time quantitative PCR analysis of periodic retained introns and total mRNAs for three
559 selected genes. Cells were synchronized by double thymidine block and samples were collected
560 0, 3, 6, 9, 12 and 15 hours post release. Errors bars represent standard deviation of the mean.
561 Diagram below indicates cell cycle stage.
562 E, Schematic representation of AURKB AS pattern. Line graph showing the relationship between
563 intron retention and mRNA levels for the AURKB gene across the cell cycle. Percent intron
564 retention (solid red line) across cell cycle was used to determine the fraction of total mRNAs
565 (solid blue line) not containing an intron, i.e. ‘corrected’ mRNA levels (dashed blue line).
566
567 Figure 2: Cell cycle-dependent regulation of CLK1.
568 A, Immunoblot analysis of proteins involved in splicing regulation in synchronized HeLa cells
569 after release from double thymidine block.
26 570 B, Immunoblot analysis of selected proteins in asynchronous HeLa cells or cells arrested at
571 different cell cycle stages. Stably expressed exogenous CLK1 levels were also assessed during
572 the cell cycle (bottom panel).
573 C, Immunoblot of endogenous CLK1 (top) and exogenously-expressed wild type (CLK1wt) or
574 kinase catalytically inactive (CLK1KD) proteins (bottom) upon treatment with 10 µM TG003.
575 D, Co-expression of CLK1WT and CLK1KD at different ratios.
576 E, Immunoprecipitation of CLK1 proteins co-expressed with myc-ubiquitin. Cells were treated
577 with 10 μM TG003 and 10 μM MG132 prior to sample collection.
578 F, Immunoblot analysis of lysates from cells synchronized upon early S phase (double
579 thymidine) release with or without TG003 treatment.
580
581 Figure 3: CLK1 regulates a network of genes that control cell cycle progression.
582 A, Identification of endogenous CLK1 targets by RNA-Seq. Numbers of different AS types
583 affected by treatment with the CLK1 inhibitor TG003 (left graph). SE, skipped exon; RI,
584 retained intron; A3E, alternative 3′ exon; A5E, alternative 5′ exon. Fraction of total analyzed
585 events that were affected by TG003 treatment (right graph).
586 B, Validation of TG003-responsive AS events by semi-quantitative RT-PCR. The bar graph
587 shows the max-delta PSI for each AS event tested in a 24-hour time course of inhibition with
588 20μM TG003.
589 C, Representation of cell cycle control genes with CLK1-dependent AS events, organized by cell
590 cycle phase and function.
591 D, Schematic representation of CHEK2 alternative splicing, showing that exon 9 encodes a
592 region overlapping the kinase domain (upper panel). Semi-quantitative RT-PCR assessment of
27 593 CHEK2 isoforms after treatment with TG003 or over-expression of the indicated factors (lower
594 left panel). RNAi of SRSF1 in cells and subsequent analysis of CHEK2 splicing by semi-
595 quantitative RT-PCR (lower right panel). PSI values are shown below gel.
596 E, Normalized CENPE total mRNA expression during an unperturbed cell cycle (triangle
597 denotes mitosis, left panel) and diagram of CENPE splicing (right). TG003-treatment of HeLa
598 cells released from G1/S arrest followed by semi-quantitative RT-PCR analysis of CENPE
599 isoforms (bar graph).
600 F, Schematic of RNA-Seq analysis of CLK1 inhibition during cell cycle (left). AS events that
601 were identified as being differentially regulated between G1 and G2 phase (top bar of bar graph)
602 and number of events that were blocked by the indicated conditions (bottom 4 bars).
603
604 Figure 4: CLK1 is required for cell cycle progression and proliferation.
605 A, Immunoblot analysis of CLK1 proteins after stable shRNA knockdown in HeLa cells.
606 Bottom, DNA content as measured by propidium iodide staining following flow cytometry.
607 B, Immunofluorescence microscopy of A549 cells depleted of CLK1 by shRNA (top row), cells
608 treated with 10 µM TG003 for 12hrs (middle row), and a control treatment with DMSO (bottom
609 row); green: tubulin, red: emerin (nuclear envelope), and blue: DAPI. Scale bar 10 µm. Right bar
610 graph shows the quantification of multinucleated cells. P values determined using Student’s t-
611 test.
612 C, Static frames from a live-cell high-content imaging movie of HeLa cells expressing Histone
613 H2B-GFP and treated with TG003 (top panel). Time after start of the experiment is indicated;
614 EP, end point (~960 min). TG003 treated cells with apparent cell division defects (indicated by
615 arrowheads in the bottom field) are shown in two independent fields.
28 616 D, Synchronized HeLa cells were treated with 20 µM TG003 at the indicated time points (0, 5,
617 and 10 hrs) and analyzed by propidium iodide staining and flow cytometry to measure DNA
618 content. Percent of 2N (lower bar graph) and 4N (upper bar graph) cells were quantified at each
619 time point as indicated in the treatment scheme (top).
620 E, Colony formation assay of HeLa cells depleted of CLK1 by shRNA, or continuously treated
621 with TG003 or KHCB-19 at the indicated concentrations.
622 F, Box plot representation of CLK1 mRNA expression levels in paired normal and tumorous
623 kidney tissue. 72 cases were analyzed.
624 G, Kaplan-Meier plot showing survival differences between patients with kidney tumors with
625 high CLK1 (red, upper quartile) or reduced CLK1 (blue, lower three quartiles) expression.
626 H, Number of cancer-associated AS events that are also regulated by CLK1 in different tumor
627 types. BRCA, Breast invasive carcinoma; COAD, Colorectal adenocarcinoma; KIRC, Kidney
628 renal clear cell carcinoma; LUAD, Lung adenocarcinoma; LUSC, Lung squamous cell
629 carcinoma; LIHC, liver hepatocellular carcinoma.
630
631
632 Legends for figure supplements
633 Figure 1 –figure supplement 1: Identification of periodic AS by multiple analysis pipelines.
634 A, Number of sequencing reads per sample (top). RNA-Seq reads and periodic seeds used for the
635 identification of all periodically expressed and spliced genes (bottom, see methods).
636 B,C, Dot plot of periodic score and false discovery rate (FDR) for each exon analyzed by the
637 MISO and VAST-TOOLS analysis pipelines. Dashed lines show FDR and periodic score cutoff
638 (see methods).
29 639 D, Heat map representation of periodically-spliced events identified by the VAST-TOOLS
640 pipeline. Data are row-normalized. Diagram below indicates cell cycle stage.
641 E, Bar graphs showing the number of periodic AS events identified separated by event type and
642 shown as a fraction of total events identified (SE: skipped exon, RI: retained intron, A3:
643 alternative 3’splice site, A5: alternative 5’splice site). MISO analysis (left panel in blue) and
644 VAST-TOOLS analysis (right panel in red).
645 F, Venn diagram representation of the overlap between periodic AS identified by VAST-TOOLS
646 and periodically expressed mRNAs (top). Venn diagram representation of the overlap between
647 periodic AS events as identified by both VAST-TOOLS and MISO (bottom, see methods).
648 G, Spearman’s rank correlation analysis of each cell cycle time point according to commonly
649 detected alternative exons by MISO and VAST-TOOLS. Spearman’s rho values are shown in
650 heat map.
651
652 Figure 2 –figure supplement 1: Periodic expression of RBPs
653 (A) Heat map representation of RNA-bound proteins (RBPs) with periodic expression. Row-
654 normalized FPKM levels are shown.
655 (B) GO analyses for the functional enrichment in the periodic RBPs.
656 (C) Number of periodic AS events that significantly correlate (Spearman’s Rho > |.75|, P < .05)
657 with the expression pattern of each RBP during cell cycle. Expression pattern of known two
658 known splicing factors, SRSF2 and ESRP2, is shown in inset.
659 (D) Average PSI values of periodic that peak at either G1 (red line) or M phase (blue line) (top
660 panel). k-mer enrichment in periodic exons as judged by Z score and separated by cell cycle
661 phase (y-axis = G1-S and x-axis = G2-M) (bottom panel).
30 662
663 Figure 2 –figure supplement 2: Regulation of CLK1 proteins levels during the cell cycle is
664 degradation-dependent.
665 A, CLK1 mRNAs as measured in synchronized cells by RNA-Seq (left) or in cells arrested at
666 each cell cycle phase, followed by quantitative RT-PCR (right).
667 B, Diagram depicting alternative the splicing pattern for CLK1 pre-mRNA (left). The short form
668 represents skipping of exon 4 that introduces a premature stop codon and is targeted by the non-
669 sense mediated decay pathway, while long form represents the full-length active isoform. Right
670 panels show levels of CLK1 variants, as measured by semi-quantitative RT-PCR with primers
671 that simultaneously detect both forms, in cells upon early S phase release or in cells arrested at
672 specific stages.
673 C, Protein stability of CLK1 is affected by its activity. Cyclohexamide chase experiment was
674 used to measure the stability of CLK1 in cells expressing either CLK1wt or the catalytic mutant
675 CLK1KD. MG132 was used to block proteasomal degradation.
676 D, Immunoprecipitation of Flag-CLK1 from cells after 3hrs of MG132 treatment and subsequent
677 detection of polyubiquitination by immonoblotting. The arrow indicates the expected position of
678 unmodified CLK1. Bottom panel shows FLAG-CLK1 protein.
679 E, HeLa cells stably expressing Flag-CLK1 were synchronized by double thymidine block in the
680 presence or absence of TG003. Phosphorylated histone 3B was detected as cell cycle marker.
681
682 Figure 3 –figure supplement 1: CLK1 regulates a network of genes that control cell cycle
683 progression.
31 684 A, Identification of endogenous CLK1 targets by RNA-Seq. Cells were treated with 10 μM
685 TG003 for 18hrs and poly-A+ RNA was sequenced. Genes significantly up-regulated or down-
686 regulated upon CLK1 inhibition (log(FPKM) are plotted, all plotted genes meet P <10-7).
687 B, Scatter plot representation of cassette exon inclusion after TG003 treatment by MISO
688 analysis. Log Bayes factor values are shown on y-axis and delta PSI on x-axis.
689 C, Scatter plot representation of retained intron levels after TG003 treatment. Log Bayes factor
690 values are shown on y-axis and delta PSI on x-axis.
691 C, Time course experiment after TG003 treatment. Cells were collected at the indicated time
692 points and the absolute delta PSI is plotted.
693 E, Immonoblot analysis of reciprocal co-immunoprecipation experiments between CHEK2
694 isoforms as indicated.
695
696 Figure 4 –figure supplement 1: Loss of CLK1 results in cell cycle defects in multiple cell
697 types.
698 A, Cell cycle composition as measured by propidium iodide staining of DNA and flow
699 cytometry analysis of cells that have been depleted of CLK1 by the indicated shRNAs.
700 B, Representative histograms of propidium iodide stained H157cells to determine cell cycle
701 defect after RNAi of CLK1.
702 C, HeLa cells treated with TG003 also have defective cell division. Immunofluorescence
703 microscopy was used to detect multi-nucleated cells, and a representative field is shown
704 (green:tubulin, red:emerin, blue:DAPI).
32 705 D, Representative histograms of DNA content in synchronous HeLa cells treated with TG003.
706 The time after early S phase release (when TG003 was added) is indicated, These data are
707 associated with Figure 4 of the main text.
708 E, Representative image from anchorage-independent growth assays (soft agar assay) of HeLa
709 cells after depletion of CLK1.
710 F, Relative mRNA expression levels of CENPE and HMMR during cell cycle. Data obtained
711 from RNA-Seq analysis. Dashed line represents 6 hours after release from G1/S.
712
713
714 Figure 4 –figure supplement 2: CLK1 mis-regulation in human cancer
715 A, Overlap of AS events that were altered in kidney cancers (as compared to normal kidney
716 samples) and AS that was altered in asynchronous cells treated with TG003 (left panel). Pie
717 chart denoting if AS in cancer occurred in the expected direction, that is, normal kidney
718 resembled TG003 treatment while tumor kidney resembled untreated cells (see methods).
719 B, Boxplot representation of CLK1, CLK2, CLK3 and CLK4 mRNA levels in 72 paired normal
720 vs. cancer kidney cancer (cRCC) samples. Kolmogorov-Smirnov test significance for each
721 factors is as follows CLK1: P = 3 × 10-5, CLK2: P = 1.2 × 10-7, CLK3: P = 2.8 × 10-12, CLK4: P
-16 722 = 2.2 × 10
723 C, Kaplan-Meier plot of kidney (cRCC) patients with tumors expressing high CLK4 (red, upper
724 quartile) vs. normal CLK4 (blue, 1-3 quartile).
725 D, PARD3 exon (chr10:34661426-34661464) PSI levels in five cancer cases are shown as an
726 example of TG003-sensitive AS which is altered between normal and cancer tissues.
727
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875
37 Dominguez Figure-1:
A 1,747 AS events in B C 1,293 genes Periodic Gene SE DNA metabolic process 1049 Expression cell cycle A3 regulation of translation 133 cell cycle process A5 posttranscriptional regulation of gene expression 1160 DNA replication Periodic AS regulation of cellular protein metabolic process RI mitotic cell cycle cell cycle phase chromatin modification response to DNA damage stimulus DNA repair RNA processing qRT-PCR validation of microtubule cytoskeleton organization periodic introns cytoskeleton organization D chromosome organization Total mRNA M phase Retained Intron macromolecule catabolic process regulation of mitotic cell cycle AURKB cellular macromolecule catabolic process microtubule-based process 3 RNA splicing cellular response to stress mRNA metabolic process 2 1x10 - 31 5x10- 2 1 (P value) n.s. G1/S S G2 M 0 E 3 GGUK1 TGA total mRNA 2 non-coding 1 coding Stage/(G1-S) 0 G1/S S G2 M Total mRNA max Retained Intron Corrected 2 HMG20B 1
1 0.5 rel. level Stage/(G1-S) 0 0 min S G2 M G1 S G2 M G1 S G1/S S G2 M M M Dominguez Figure-2:
ACB E Hours post G1/S release 0 1 3 5 7 9 12 15 18 TG003: CCNB1 -+ Flag-CLK1 Clk1 CLK1
Vector Vector WT KD CLK1 TUB1A Clk2 TG003: - - + - + TG003: -+ -+ CLK2 SRPK1 Flag-CLK1 SRPK1 SRSF1 TUB1A Clk Clk wt KD myc-Ub SRSF1 CCNB1 D IP: Flag endogenous proteins TUB1A CLK1 : + ++ +++ HNRNPA0 wt Flag CLK1KD : + + + Flag-CLK1 Flag-CLK1 wt Input Flag HNRNPA1 TUB1A HA-CLK1KD Flag-CLK1 HNRNPA2 TUB1A
HNRNPA3
DAZAP1 F G1/S S G2 M G1/S S G2 M HNRNPF 0 1 3 5 7 9 12 151 18 0 1 3 5 7 9 12 151 18 :hrs post G1/S HNRNPL CLK1 CCNB1 RBM4 SRSF1 PTBP1 TUB1A G1/S S G2 M DMSO TG003 Dominguez Figure-3:
A B Fraction of No. of events annotated events TG003 DMSO 100 600 400 200 0 0 0.02 0.04 DMSO RNA-seq SE 50 .vs PSI PSI increase RI 0 PSI decrease A3E TG003 A5E C
DNA repair centriole metaphase & replication RAD51AP1 CHDL1 duplication anaphase NASP1 SMC6 CEP120 kinetochore CDK1 cytokinesis chromosome altered cell cycle REPIN1 XRCC6 TDP1 CDK5RAP2 CLASP1 CENPK MLF1 SEPT10
by CLK1 G1/S condensation entry/arrest transition RFC2 CHEK2 * EXO1 CEP68 KATNA1 SASS6 NUF2 ANLN MDM1 CCNE NPAT FANCA RIF1 CEP290 ADD3 INCENP CDC25C CENPE* SEPT6 RB1 UHRF2 CDK7 MRE11A RAD1 CEP70 RCC1 CENPN SEPT2 G1 S G2 mitosis CCND CCNE CCNA CCNB classical
regulators CDK4/6 CDK2 CDK1
D CHEK2 pre-mRNA CHK2 FL E CENPE total mRNA CHK2 ∆9 (kinase domain CENPE pre-mRNA destroyed) Rel. Kinase Domain
SCD FHA expression DMSO TG003 100% short RNAi: 50%
TG003 PSI long - + ++ 0% Hrs. G1/S: 0 3 6 9 12 15 0 3 6 9 12 15 CHK2 FL CHK2 FL
CHK2 ∆9 CHK2 ∆9 CENPE PSI: 72% 80% 92% 92% 93% 69%88%63% 76% 91% SRPK1 α-SRSF1 Hrs. G1/S: 0 3 6 9 12 15 18 0 3 6 9 12 15 18 α-CLK1
proteins DMSO TG003 F
Cell synchrony and release into CLK1 No. of altered AS events inhibitors TG003 G1 (t=0) vs. G2 (t=6)
G1 DMSO Blocked by TG 81%
KHCB19 Blocked by KH 78%
Blocked by either 94%
t = 0 (G1/S) t = 6 (early G2) Blocked by Both 65%
Dominguez Figure-4:
A CLK1 sh B tubulin emerin DAPI merge p=.005 CLK1 p=.0002 TUB1A 50 n=316 shCLK1
ctl sh CLK1 sh3 n=102 25 TG003 n=616 multi-nucleated
% 0 DMSO C TG003 DMSO
G1/S DErelease F
G1/S G2 M G1 S G2 M CLK1 mRNA P = 1×10-5 0 3 6 9 12 15
TG: 3000 Ctl shRNA CLK1 shRNA1 CLK1 shRNA2 100 CTRL TG0 75 TG 5 2000 50 TG10 25 % of 4N cells 0 DMSO TG 20 µM TG 40 µM 1000 100 6 9 12 15 rel. expression 75 50 25
% of 2N cells 0 6 9 12 15 DMSO KHCB 20 µM KHCB 40 µM Hours post G1/S G H median survival (years) CLK1-regulated AS events with high 4.45 cancer-specific alteration normal 7.57 1 P = .007 100 .8 75 .6 50 .4 25
.2 No. of Events survival probability CLK1 mRNA 0 0 0 2 4 6 8 10 Time (years) Tumor type